Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation
Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation
复制标题
从中国工业黄酒酵母中分离单倍体用于代谢工程操作
DOI：
10.1002/jib.97
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发表时间：
2013-12
影响因子：
2.6
通讯作者：
Xie, Guangfa
中科院分区：
文献类型：
--
作者：
Shen, Chao;Lu, Jian;Chen, Jian;Xie, Guangfa
酵母菌复杂的代谢过程影响着葡萄酒的发酵，从而影响着葡萄酒的质量。葡萄酒酵母，由于它们是典型的原养型和多倍体，已被限制在利用经典的重组遗传技术来改善其特性。为了克服这个问题，已经通过破坏HO基因从商业化的中国米酒菌株N85中分离出了单倍体。在本研究中，使用Cre-loxP系统和可去除的G418 r标记来构建HO破坏盒。构建的破坏盒的大部分异源序列通过KanMX基因的环出（loop-out），通过Cre重组酶的诱导表达，成功地从单倍体的基因组中切除。抗性标记的去除确保了菌株的生物安全性。正如预期的那样，在亲本和单倍体菌株之间没有观察到发酵能力的差异。本工作报告了一个HO中断盒的降落聚合酶链反应的构建和应用与中国米酒酵母的单倍体分离，扩大生理研究和工业应用。版权所有© 2013酿酒和蒸馏研究所
The complex metabolic processes of yeast influence wine fermentation and therefore the quality of wine. Wine yeasts, owing to their being typically prototrophic and often polyploid, have been restricted in terms of exploiting classical recombinant genetic techniques to improve their characteristics. To overcome this problem, haploids have been isolated from a commercial Chinese rice wine strain N85, by disruption of the HO gene. In this study, the Cre–loxP system and a removable G418r marker were used to construct an HO disruption cassette. Most of the heterologous sequences of constructed disruption cassette were successfully excised from the genome of the haploids by loop-out of the KanMX gene, through induced expression of the Cre recombinase. The removal of the resistant marker ensures the biological safety of the strains. As expected, no difference in fermentation capacity between the parental and the haploid strains was seen. The present work reports the construction of an HO disruption cassette by touchdown polymerase chain reaction and its application with a Chinese rice wine yeast for haploid isolation and to broaden physiological investigations and industrial applications. Copyright © 2013 The Institute of Brewing & Distilling
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